A wide variety of proteins require post-translational prenylation to participate normally in various cellular processes. For example, proteins such as Ras must become prenylated for proper membrane localization and activity. See e.g., Adjei, (2003) Lung Cancer 41 Suppl 1, S55-62; Rowinsky, et at. (1999) J Clin Oncol 17, 3631-52; Zhu, et al., and Sebti, S. M. (2003) Curr Opin Investig Drugs 4, 1428-35; Zhang, et al. (1996) Annu Rev Biochem 65, 241-69; Vergnes, et al. (2004) Proc Natl Acad Sci U S A 101, 10428-33.
Protein prenylation includes protein farnesylation, which involves the transfer of a farnesyl moiety to a protein and protein geranylgeranylation, which involves the transfer of a geranylgeranyl moiety to a protein. Farnesyl diphosphate (FPP) is the natural substrate of farnesyl transferase (FTase), which catalyzes the transfer of a farnesyl moiety from FPP to proteins. A group of compounds known as FTase inhibitors (FTIs) have the ability to block protein farnesylation. Similarly, geranylgeranyl diphosphate (GGPP) is the natural substrate of geranylgeranyl transferase I (GGTaseI), which catalyzes the transfer of a geranylgeranyl moiety from GGPP to proteins. A group of compounds known as GGTaseI inhibitors have the ability to block protein geranylgeranylation.
FTase catalyzes the farnesylation, and GGTaseI catalyzes the geranylgeranylation, of proteins with a cysteine residue located in a C-terminal Ca1Ca2X motif, where C is the modified cysteine, a1 and a2 are often an aliphatic residue, and X is Ser, Met, Ala, or Gln. See Roskoski, R., Jr. (2003) Biochem Biophys Res Commun 303, 1-7; Dunten, et al. (1998) Biochemistry 37, 7907-7912; Caplin, et al. (1998) J Biol Chem 273, 9472-9.
As mentioned above, prenylation of proteins, such as Ras, is often required for proper participation of the protein in various cellular processes. Specifically, prenylation is obligatory for the oncogenic effects of mutant Ras. See Adjei, (2001) J Natl Cancer Inst 93,1062-74. Mutated forms of Ras genes are among the most common genetic abnormality in human cancer, occurring in 10 to 30% of all neoplasms. See Anwar, et al. (1992) Cancer Res 52, 5991-6; Watanabe, et al. (1994) Int J Cancer 58, 174-8; Konishi, et al. (1995) Am J Pathol 147, 1112-22; Konishi, et al. (1997) Prostate 30, 53-7. These observations have lead to the development of a number of FTIs, which block protein farnesylation, and GGTaseI Inibitors, which block geranylgeranylation. FTIs, for example, have the ability to inhibit Ras farnesylation, appropriate subcellular localization and activity, in addition to inhibiting growth of Ras-transformed cells. Various FTIs are being studied for their efficacy as antineoplastic agents. See Santucci, et al. (2003) Cancer Control 10, 384-7; Sebti, et al. (2000) Oncogene 19, 6584-93; Reid, T. S., and Beese, L. S. (2004) Biochemistry 43, 6877-84.
Prenylation is the first and obligatory step in a series of post-translational modifications which mediate membrane localization and possibly protein-protein interactions for a variety of proteins involved in cellular regulatory events. See Ramamurthy, et al. (2003) Proc Natl Acad Sci U S A 100, 12630-5; Scheffzek, et al. (2000) Nat. Struc. Biol. 7, 122-126; Chen, et al. (1999) Chin Med Sci J 14, 138-44. Subsequent to farnesylation, the a1a2X peptide is cleaved by the endopeptidase RCE1 followed by carboxymethylation of the now terminal prenylated cysteine residue by the carboxymethyltransferase Icmt. See Bergo, et al. (2002) Mol Cell Biol 22, 171-81; Michaelson, et al (2005) Mol Biol Cell. 
Similar post-translational modifications occur on the relatively small set of farnesylated cellular proteins, not all of which have been identified or characterized. Therefore, the rapid and selective detection of cellular protein prenylation status and easy isolation of farnesylated proteins would be helpful to understanding both the function of farnesylated proteins and of FTase inhibitors. Similar post-translational modifications also occur on a set of geranylgeranylated cellular proteins, and the rapid and selective detection of cellular protein prenylation status and easy isolation of geranylgeranylated proteins would be helpful to understanding both the function of geranylgeranylated proteins and of GGTaseI inhibitors.
Monitoring the prenylation status of proteins in cells is a challenging undertaking. Initially, prenylation of proteins was discovered by metabolic labeling with [3H]mevalonolactone. See Maltese (1990) Faseb J 4, 3319-28. In these experiments, some of the tritium label was incorporated into both farnesylated and geranylgeranylated proteins. Subsequently, a salvage pathway was discovered where radiolabeled farnesol (FOH) and geranylgeraniol (GGOH), precursors of FPP and GGPP, were selectively incorporated into their respective farnesylated or geranylgeranylated proteins. See, e.g., Andres, et al. (1999) Methods Mol Biol 116, 107-23. A drawback of these approaches is the inherently low sensitivity of autoraidographic detection of the weak tritium β-emission. In fact, it can take up to four weeks to visualize proteins extracted from cells labeled with tritiated mevalonate or farnesol by audioradiography. See Gibbs, et al. (1999) J. Med. Chem. 42, 3800-3808. Also, the incorporation of tritiated prenyl groups does not provide a convenient method for isolation of these modified proteins.
The facile detection, isolation and purification of prenylated proteins based solely on their post-translational modification status is useful for developing an understanding of the mechanism of cellular growth inhibition by inhibitors of enzymes catalyzing prenylation reactions (e.g., FTIs, GGTaseI Inhibitors).
Antibodies are useful both in the routine detection and immunoprecipitation of proteins with other post-translational modifications, such as phosphorylation. See Gronborg, et al. (2002) Mol Cell Proteomics 1, 517-27. However, reports of previous attempts to produce antibodies to detect farnesylated proteins or geranylgeranylated proteins have not had overwhelming success. For example, two of these reports described non-specific antibodies that could not differentiate between proteins modified by farnesylation, geranylgeranylation or other lipids. See Liu, et al. (2004) Bioconjug Chem 15, 270-7; Lin, et al. (1999) J Gen Virol 80 (Pt 1), 91-6. The other report from Baron et al. does describe the production of anti-farnesyl antibodies specific for farnesylation, but the analysis for specificity was limited. Baron, et al. (2000) Proc Natl Acad Sci U S A 97, 11626-31. All of the commercially available sources for anti-farnesyl antibodies cross-react with geranylgeranylated proteins.
Several unnatural analogs of FPP appear to be utilized by cells and incorporated into the proteins by FTase, including the unnatural FPP analogue 3-vinyl-farenesol, a pro-drug of the FTase transferable 3-vinyl-farnesyl diphosphate (3vFPP), and 8-azido-farnesol. A tagging-via-substrate (TAS) approach to the detection and isolation of farnesylated proteins involving the incorporation of 8-azido-farnesol into cellular proteins has been developed by Kho, et al. (2004) Proc Natl Acad Sci U S A 101, 12479-84. By this approach, modified proteins are isolated from cell lysates using a biotinylated phosphine capture reagent and subsequently identified by mass spectrometry. However, the sensitivity is relatively low and the technique does not lend itself to the routine detection of modified proteins.
Accordingly, there remains a need in the art for a method and system for convenient, rapid, and selective detection, identification, and isolation of prenylated proteins, including farnesylated proteins and geranylgeranylated proteins, which satisfactorily address the above-identified problems.